Barrett's esophagus is a pre-neoplastic disease that predisposes patients to development of esophageal adenocarcinoma (EAC). Approximately 3 million people in the US have Barrett's esophagus and they are frequently screened by endoscopy with pathological analyses of esophageal biopsies. The goal of the screening is to detect premalignant changes early to enable interventions to prevent EAC. However, the pathology analyses are subjective and prone to variation and cannot predict which patients will develop EAC. There is an unmet need for accurate prognostic tests to identify high risk patients who require therapeutic interventions to prevent EAC and to identify low risk patients who may be spared unnecessary, costly therapy and surveillance. Such tests are key to reducing EAC incidence and improving the efficiency of health care spending in this disease area. The broad objective of the proposed Phase II study is to validate a prognostic test, termed TissueCypher, which predicts future risk of developing high grade dysplasia (HGD) and EAC in patients with Barrett's esophagus. The technological innovation of the proposed study is Cernostics' TissueCypher technology, a systems biology-based approach to anatomic pathology. The technology employs multiplexed fluorescence labeling of key tissue system biomarkers, including malignant, immune and stromal processes in tissue specimens, with whole slide fluorescence imaging and image analysis to quantify biomarkers in the context of tissue morphology. This is coupled to multivariate classifiers to integrate biomarker data with clinical variables to produce diagnostic, prognostic and predictive scores. The technical and scientific merit and feasibility of the TissueCypher technology has been demonstrated in Phase I stage-type of research through other funding sources. Preliminary studies have evaluated 14 biomarkers and morphology in a multi- institution cohort of patients with Barrett's esophagus. An optimal set of 6 biomarkers, 15 image analysis measurements derived from the biomarkers and morphology and a multivariate classifier have been selected, which form the basis of the TissueCypher Barrett's risk assessment test that will be validated in the Phase II study. The specific aims of the Phase II study are to develop the final format of the TissueCypher test and perform the analytical and clinical performance studies that are required for commercialization. The consortium of investigators at Cernostics, University of Pittsburgh and Cleveland Clinic will reconfigure the test format and perform analytical and independent clinical validation studies on the TissueCypher test towards the goal of offering the test as a laboratory-developed test (LDT) via a CLIA-certified lab. The test will be offered as a service to gastroenterologists and pathologists to enable accurate, individualized risk stratification for Barrett's patients to improe disease management and reduce the incidence of EAC.